U.S. patent number 6,423,696 [Application Number 09/549,793] was granted by the patent office on 2002-07-23 for inhibition of arylamine n-acetyl transferase.
This patent grant is currently assigned to The United States of America, as represented by the Department of Health &. Invention is credited to Jerry M. Collins, Aspandiar G. Katki, Raymond W. Klecker.
United States Patent |
6,423,696 |
Collins , et al. |
July 23, 2002 |
Inhibition of arylamine N-acetyl transferase
Abstract
The present invention is directed to a method of inhibiting
arylamine N-acetyl transferase (NAT) from acetylating an arylamine
group in a substrate. The method comprises contacting NAT with an
inhibitor that interacts with NAT and thereby inhibits NAT from
acetylating said arylamine group in said substrate. Preferably, NAT
is in vivo, such as in a mammal, and the substrate is a drug.
Preferably, the method inhibits acetylation of arylamine substrates
which are inhaled, ingested, or absorbed through the skin, wherein
acetylation of the substrate predisposes a mammal to a biological
disorder or a disease. The present invention also provides a
composition comprising a compound comprising an arylamine group and
an inhibitor, wherein the inhibitor interacts with NAT to inhibit
NAT from acetylating the arylamine group in the compound.
Inventors: |
Collins; Jerry M. (Rockville,
MD), Klecker; Raymond W. (Silver Spring, MD), Katki;
Aspandiar G. (Gaithersburg, MD) |
Assignee: |
The United States of America, as
represented by the Department of Health & (N/A)
(Washington, DC)
|
Family
ID: |
26827829 |
Appl.
No.: |
09/549,793 |
Filed: |
April 14, 2000 |
Current U.S.
Class: |
514/159 |
Current CPC
Class: |
A61K
31/136 (20130101); A61K 31/18 (20130101); A61K
31/4409 (20130101); A61K 31/505 (20130101); A61K
31/60 (20130101); A61K 31/606 (20130101); A61K
31/60 (20130101); A61K 2300/00 (20130101) |
Current International
Class: |
A61K
31/60 (20060101); A61K 31/136 (20060101); A61K
31/18 (20060101); A61K 31/4409 (20060101); A61K
31/505 (20060101); A61K 031/60 () |
Field of
Search: |
;514/159 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Lutoslawska, "Isoniazid metabolism in the small intestine wall of
rats", Acta Pol. Pharm. (1981), 38(6), pp. 711-715, abstract.*
.
Cribb et al., "Expression of Monomorphic Arylamine
N-Acetyltransferase (NAT1) in Human Leukocytes," Journal of
Pharmacology and Experimental Therapeutics, vol. 259, No. 3, pp.
1241-1246 (1991). .
Cribb et al., "Role of Polymorphic and Monomorphic Human Arylamine
N-Acetyltransferases in Determining Sulfamethoxazole Metabolism,"
Biochemical Pharmacology, vol. 45, No. 6, pp. 1277-1282 (1993).
.
Frederickson et al., "Relationship between in Vivo Acetylator
Phenotypes and Cytosolic N-Acetyltransferase and
O-Acetyltransferase Activities in Human Uroepithelial Cells,"
Cancer Epidemiology Biomarkers and Prevention, vol. 3, No. 1, pp.
25-32 (1994). .
Lang, "Acetylation as an Indicator of Risk," Environmental Health
Perspectives, vol. 105, Supplement 4, pp. 763-766 (Jun. 1997).
.
Lo et al., "The Effect of Sulinadac on Arylamine
N-acetyl-transferase activity in Pseudomonas aeruginosa," Microbios
vol. 93, pp. 159-168 (1998)..
|
Primary Examiner: Weddington; Kevin E.
Attorney, Agent or Firm: Leydig, Voit & Mayer, Ltd.
Parent Case Text
This application claims priority to U.S. provisional patent
application Serial No. 60/129,687, filed Apr. 16, 1999.
Claims
What is claimed is:
1. A method of inhibiting arylamine N-acetyl transferase (NAT) from
acetylating an arylamine group in a substrate in vivo, which method
comprises contacting NAT with an inhibitor that interacts with NAT
in vivo and thereby inhibits NAT from acetylating said arylamine
group in said substrate.
2. The method of claim 1, wherein NAT is in a mammal.
3. The method of claim 1, wherein said inhibitor comprises an
arylamine group that can be acetylated by NAT.
4. The method of claim 1, wherein said inhibitor is a compound of
formula: ##STR3## wherein one or more carbon atoms at positions 2,
3, 5 and 6 can be heteroatoms, which can be the same or different,
wherein R.sub.1 is one or more substituents, which can be the same
or different, selected from the group consisting of hydrogen,
hydroxy, an alkoxy, sulfhydryl, nitro, amino, a halo, an aryloxy,
cyano, --[SO.sub.2 --R.sub.4 ], an alkyl, a cycloalkyl, a
heterocycloalkyl, an alkenyl, an alkynyl, an aryl, a heteroaryl, an
arylalkyl and a heteroarylalkyl, wherein R.sub.2 is a substituent
selected from the group consisting of amino, carboxyamide,
sulfamide, --[NH--NH.sub.2 ], --[SO.sub.2 --NH--NH.sub.2 ] and
--[CO--NH--NH.sub.2 ], wherein R.sub.3 is a substituent selected
from the group consisting of hydrogen, hydroxy, an alkoxy,
sulfhydryl, nitro, amino, a halo, an aryloxy, cyano, --[SO.sub.2
--R.sub.4 ], an alkyl, a cycloalkyl, a heterocycloalkyl, an
alkenyl, an alkynyl, an aryl, a heteroaryl, an arylalkyl and a
heteroarylalkyl, wherein R.sub.4 is a substituent selected from the
group consisting of hydrogen, hydroxy, an alkoxy, sulfhydryl,
nitro, amino, a halo, an aryloxy, cyano, an alkyl, a cycloalkyl, a
heterocycloalkyl, an alkenyl, an alkynyl, an aryl, a heteroaryl, an
arylalkyl, a heteroarylalkyl, and NH--R.sub.5, wherein R.sub.5 is a
substituent selected from the group consisting of hydrogen,
hydroxy, an alkoxy, sulfhydryl, nitro, amino, a halo, an aryloxy,
cyano, an alkyl, a cycloalkyl, a heterocycloalkyl, an alkenyl, an
alkynyl, an aryl, a heteroaryl, an arylalkyl and a heteroarylalkyl,
wherein said substituent is unsubstituted or substituted.
5. The method of claim 4, wherein said heteroatoms are selected
from the group consisting of oxygen, nitrogen and sulfur.
6. The method of claim 4, wherein said substituent is substituted
with one to three groups, which can be the same or different and
are selected from the group consisting of an alkyl, an alkenyl, an
alkynyl, .dbd.O, a halo, hydroxy, a lower alkoxy, carboxy, a
carboalkoxy, a carboxamido, cyano, carbonyl, --NO.sub.2, an
alkylthio, sulfoxide, sulfone, acylamino, amidino, an aryl, a
heteroaryl, an aryloxy, and a heteroaryloxy.
7. The method of claim 1, wherein said NAT is NAT-1 and said
inhibitor is selected from the group consisting of
para-amino-salicylate (PAS) and para-amino-benzoic acid (PABA).
8. The method of claim 1, wherein said NAT is NAT-2 and said
inhibitor is selected from the group consisting of
dichlorphenamide, sulphamethazine (SMZ), dapsone (DDS) and
isoniazid (INH).
9. The method of claim 2, wherein acetylation of said substrate
predisposes the mammal to a disease.
10. The method of claim 9, wherein said inhibitor is administered
prophylactically to inhibit acetylation of said substrate.
11. The method of claim 4, wherein NAT is in a mammal and
acetylation of said substrate predisposes the mammal to a
disease.
12. The method of claim 11, wherein said inhibitor is administered
prophylactically to inhibit acetylation of said substrate.
13. The method of claim 1, wherein said substrate is a drug.
14. The method of claim 13, wherein said drug is an anticancer
drug, an aminosalicylate, sulfasalazine, or a sulfonamide.
15. The method of claim 13, wherein acetylation of said drug
adversely affects its therapeutic or prophylactic effect.
16. The method of claim 4, wherein said substrate is a drug.
17. The method of claim 16, wherein said drug is an anticancer
drug, an aminosalicylate, sulfasalazine, or a sulfonamide.
18. The method of claim 16 wherein acetylation of said drug
adversely affects its therapeutic or prophylactic effect.
19. The method of claim 1, wherein said substrate and said
inhibitor are co-administered.
20. The method of claim 13, wherein said substrate and said
inhibitor are co-administered.
21. The method of claim 16, wherein said substrate and said
inhibitor are co-administered.
22. A pharmaceutical composition comprising: (i) a compound
comprising an arylamine group, (ii) an inhibitor, wherein said
inhibitor interacts with NAT to inhibit NAT from acetylating said
arylamine group in said compound and wherein said inhibitor is
present in said composition in an amount sufficient to inhibit
acetylation of the arylamine group in the compound, and (iii) a
pharmaceutically acceptable carrier.
23. The composition of claim 22, wherein said inhibitor comprises
an arylamine group that can be acetylated by NAT.
24. The composition of claim 22, wherein said inhibitor is a
compound of formula: ##STR4## wherein one or more carbon atoms at
positions 2, 3, 5 or 6 can be heteroatoms, which can be the same or
different, wherein R.sub.1 is one or more substituents, which can
be the same or different, selected from the group consisting of
hydrogen, hydroxy, an alkoxy, sulfhydryl, nitro, amino, a halo, an
aryloxy, cyano, --[SO.sub.2 --R4], an alkyl, a cycloalkyl, a
heterocycloalkyl, an alkenyl, an alkynyl, an aryl, a heteroaryl, an
arylalkyl and a heteroarylalkyl, wherein R.sub.2 is a substituent
selected from the group consisting of amino, carboxyamide,
sulfamide, --[NH--NH.sub.2 ], --[SO.sub.2 --NH--NH.sub.2 ] and
--[CO--NH--NH.sub.2 ], wherein R.sub.3 is a substituent selected
from the group consisting of hydrogen, hydroxy, an alkoxy,
sulfhydryl, nitro, amino, a halo, an aryloxy, cyano, --[SO.sub.2
--R.sub.4 ], an alkyl, a cycloalkyl, a heterocycloalkyl, an
alkenyl, an alkynyl, an aryl, a heteroaryl, an arylalkyl and a
heteroarylalkyl, wherein R.sub.4 is a substituent selected from the
group consisting of hydrogen, hydroxy, an alkoxy, sulfhydryl,
nitro, amino, a halo, an aryloxy, cyano, an alkyl, a cycloalkyl, a
heterocycloalkyl, an alkenyl, an alkynyl, an aryl, a heteroaryl, an
arylalkyl, a heteroarylalkyl, and NH--R.sub.5, wherein R.sub.5 is a
substituent selected from the group consisting of hydrogen,
hydroxy, an alkoxy, sulfhydryl, nitro, amino, a halo, an aryloxy,
cyano, an alkyl, a cycloalkyl, a heterocycloalkyl, an alkenyl, an
alkynyl, an aryl, a heteroaryl, an arylalkyl and a heteroarylalkyl,
wherein said substituent is unsubstituted or substituted.
25. The composition of claim 24, wherein said heteroatoms are
selected from the group consisting of oxygen, nitrogen and
sulfur.
26. The composition of claim 24, wherein said substituent is
substituted with one to three groups, which can be the same or
different and are selected from the group consisting of an alkyl,
an alkenyl, an alkynyl, .dbd.O, a halo, hydroxy, a lower alkoxy,
carboxy, a carboalkoxy, a carboxamido, cyano, carbonyl, --NO.sub.2,
an alkylthio, sulfoxide, sulfone, acylamino, amidino, an aryl, a
heteroaryl, an aryloxy and a heteroaryloxy.
27. The composition of claim 22, wherein said inhibitor is PAS,
PABA, dichlorphenamide, SMZ, DDS or INH.
28. The composition of claim 22, wherein said compound is an
aminosalicylate, sulfasalazine, a sulfonamide or an anticancer
drug.
29. The composition of claim 28, wherein said anticancer drug is
aminoglutethimide, amonafide or batracylin.
30. The composition of claim 24, wherein said compound is an
aminosalicylate, sulfasalazine, a sulfonamide or an anticancer
drug.
31. The composition of claim 30, wherein said anticancer drug is
aminoglutethimide, amonafide or batracylin.
32. The method of claim 2, wherein the mammal is a human.
33. The method of claim 2, wherein said substrate is a
procarcinogen.
34. The method of claim 4, wherein NAT is in a mammal and wherein
said substrate is a procarcinogen.
35. The method of claim 2, wherein acetylation of said substrate
results in the formation of a carcinogenic product.
36. The method of claim 4, wherein acetylation of said substrate
results in the formation of a carcinogenic product.
Description
TECHNICAL FIELD OF THE INVENTION
The present invention relates to the inhibition of acetylation of a
substrate by arylamine N-acetyl transferase.
BACKGROUND OF THE INVENTION
The majority of drugs are metabolized by enzymes in the body. Since
many patients are simultaneously treated with two or more drugs,
there is always the potential that one drug may interfere with the
metabolism of another drug, e.g., one may inhibit the enzyme which
transforms another. Inhibition of the metabolism of one drug by
another drug is normally problematic. Metabolic drug-drug
interactions can produce adverse reactions and nearly always
require adjustments of doses. Such situations are nuisances and are
desirably avoided.
Information on drug metabolism varies from one class of enzymes to
another. For some drug-metabolizing enzymes (e.g., the cytochrome
P450), literature on drug--drug interactions is voluminous. In
contrast, very little has been reported for the arylamine N-acetyl
transferases (NAT), i.e., enzymes which acetylate arylamine
groups.
Unlike the wide variety of forms for cytochrome P450 and
UDP-glucuronyl transferases, there are only two forms of human
arylamine N-acetyl transferase, namely, NAT-1 and NAT-2. The level
of expression varies substantially among individuals for both NAT-1
and NAT-2. NAT-2 is strongly polymorphic; indeed, it was the first
enzyme for which polymorphism in human drug metabolism was
appreciated. Among various ethnic populations, 30-80% of
individuals are "fast" acetylators via NAT-2, and the remainder are
"slow" acetylators. The polymorphism of NAT-1 has only recently
been appreciated.
Others have described N-acetyl transferases which do not play a
role in enzyme metabolism. For example, Hillman et al. (U.S. Pat.
No. 5,795,724) disclose an N-acetyl transferase (NACTH) responsible
for histone acetylation. NACTH differs from NAT in biological
function and location in the cell. NACTH acts upon an entirely
different family of substrates than NAT. In addition, NACTH is
found in the nucleus of the cell, whereas NAT is located in the
cytoplasm.
A number of compounds are known in the art which have arylamine
groups that can be acetylated by NAT. For example,
para-amino-benzoic acid (PABA), para-amino-salicylate (PAS) and
sulfamethoxazole (SMX) are acetylated by NAT-1. PABA is well-known
as a topical sunscreen. PAS is used for the therapy of
tuberculosis, but even with the recent increase in tuberculosis,
incidence of tuberculosis is still rare, and there are other more
active drugs. SMX is commonly prescribed for the treatment of
infections in the urinary tract and elsewhere. Drugs identified as
being acetylated by NAT-2 are numerous and include isoniazid (INH),
dapsone (DDS), sulphamethazine (SMZ), aminoglutethimide (AG),
procainamide, and hydralazine.
Occasionally, situations arise in which it is desirable to inhibit
the activity of a drug-metabolizing enzyme. Such situations include
stretching a scarce or expensive supply of drugs (e.g.,
cyclosporin/ketoconazole), prolonging the half-life of an active
drug to reduce the frequency of administration (e.g.,
AZT/probenecid), and blocking formation of a toxic metabolite
(e.g., in cases of methanol poisoning, the use of ethanol to
prevent formation of formaldehyde as a metabolite of methanol).
Oftentimes for NAT, it is desirable to inhibit the formation of a
toxic metabolite. In some cases, the acetylated metabolite
generated by NAT has been shown definitively to be more toxic than
the parent drug. In other cases, there is not a direct link to a
specific metabolite, but there is a strong association between high
rates of acetylation and adverse reactions. As an extension of this
category, even in the absence of drug therapy, there have been
several linkages reported between acetylation rates and
predisposition to diseases such as cancer. In certain situations,
individuals with rapid acetylation have a 10-fold (or greater) risk
of disease than individuals with slow acetylation (Lang et al.
Environmental Health Perspectives 105(suppl. 4): 763-766 (1997)).
For these situations, inhibition of NAT would be beneficial to
patients.
Therefore, there exists a need for a method of inhibiting the
acetylation of a substrate by NAT. The present invention seeks to
provide such a method, as well as a composition for use in such a
method. These and other objects and advantages of the present
invention, as well as additional inventive features, will be
apparent from the description of the invention provided herein.
BRIEF SUMMARY OF THE INVENTION
The present invention is directed to a method of inhibiting
arylamine N-acetyl transferase (NAT) from acetylating an arylamine
group in a substrate. The method comprises contacting NAT with an
inhibitor that interacts with NAT and thereby inhibits NAT from
acetylating the arylamine group in the substrate. Preferably, the
inhibitor of NAT is a compound of formula: ##STR1##
wherein one or more carbon atoms at positions 2, 3, 5 and 6 can be
heteroatoms, which can be the same or different and can be selected
from the group consisting of oxygen, nitrogen and sulfur, R.sub.1
is one or more substituents, which can be the same or different,
selected from the group consisting of hydrogen, hydroxy, an alkoxy,
sulfhydryl, nitro, amino, a halo, an aryloxy, cyano, --[SO.sub.2
--R.sub.4 ], an alkyl, a cycloalkyl, a heterocycloalkyl, an
alkenyl, an alkynyl, an aryl, a heteroaryl, an arylalkyl and a
heteroarylalkyl, R.sub.2 is a substituent selected from the group
consisting of amino, carboxyamide, sulfamide, --[NH--NH.sub.2 ],
--[SO.sub.2 --NH--NH.sub.2 ] and --[CO--NH--NH.sub.2 ], and R.sub.3
is a substituent selected from the group consisting of hydrogen,
hydroxy, an alkoxy, sulfhydryl, nitro, amino, a halo, an aryloxy,
cyano, --[SO.sub.2 --R.sub.4 ], an alkyl, a cycloalkyl, a
heterocycloalkyl, an alkenyl, an alkynyl, an aryl, a heteroaryl, an
arylalkyl, a heteroarylalkyl, wherein R.sub.4 is a substituent
selected from the group consisting of hydrogen, hydroxy, an alkoxy,
sulfhydryl, nitro, amino, a halo, an aryloxy, cyano, an alkyl, a
cycloalkyl, a heterocycloalkyl, an alkenyl, an alkynyl, an aryl, a
heteroaryl, an arylalkyl, a heteroarylalkyl, and NH--R.sub.5,
wherein R.sub.5 is a substituent selected from the group consisting
of hydrogen, hydroxy, an alkoxy, sulfhydryl, nitro, amino, a halo,
an aryloxy, cyano, an alkyl, a cycloalkyl, a heterocycloalkyl, an
alkenyl, an alkynyl, an aryl, a heteroaryl, an arylalkyl and a
heteroarylalkyl, wherein the substituent is unsubstituted or
substituted with one to three groups, which may be the same or
different and are selected from the group consisting of an alkyl,
an alkenyl, an alkynyl, .dbd.O, a halo, hydroxy, a lower alkoxy,
carboxy, a carboalkoxy, a carboxamido, cyano, carbonyl, --NO.sub.2,
an alkylthio, sulfoxide, sulfone, acylamino, amidino, an aryl, a
heteroaryl, an aryloxy, and a heteroaryloxy.
The present invention also provides a composition comprising a
compound comprising an arylamine group that can be acetylated by
NAT and an inhibitor which interacts with NAT to inhibit NAT from
acetylating the arylamine group in the compound.
The invention may best be understood with reference to the
accompanying drawings and in the following detailed description of
the preferred embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph of absorbance at 268 nm vs. time (minutes).
FIG. 2 is a graph of plasma concentration of Ac-SMX (mM) vs. time
(hours).
FIG. 3 is a graph of urine concentration of Ac-SMX (mM) vs. time
(hours).
FIG. 4 is a graph of plasma concentration of SMX (mM) vs. time
(hours).
FIG. 5 is a graph of plasma concentration of PAS (mM) vs. time
(hours).
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention provides a method of inhibiting arylamine
N-acetyl transferase (NAT) from acetylating an arylamine group in a
substrate. The method comprises contacting NAT with an inhibitor
that interacts with NAT and thereby inhibits NAT from acetylating
the arylamine group in the substrate. Preferably, NAT is in vivo.
More preferably, NAT is in a mammal, most preferably a human.
Any inhibitor of NAT can be used in the methods of the present
invention as long as it is safe and efficacious. By "inhibitor" is
meant any agent that inhibits NAT from acetylating an arylamine
group in a substrate. While desirably an inhibitor effects complete
inhibition, one of ordinary skill in the art will appreciate that
less than complete inhibition is beneficial in the context of the
present inventive method. In this regard, an inhibitor can inhibit
NAT by binding NAT in such a place and in such a manner as to
affect adversely NAT's ability to acetylate a substrate. For
example, an inhibitor can bind NAT at or near the active site of
the enzyme such that NAT is unable to bind to or acetylate a
substrate. One of ordinary skill in the art will appreciate that
binding of an inhibitor at the active site will interfere with the
ability of NAT to bind a substrate and that binding of an inhibitor
near the active site can effect a conformational change at or near
the active site such that NAT cannot bind or acetylate a substrate.
Examples of such inhibitors include immunological reagents, such as
antibodies, e.g., monoclonal and polyclonal antibodies and
immunologically reactive fragments thereof. The generation of
immunological reagents is within the skill in the art.
Alternatively, the inhibitor is, itself, a substrate of NAT, in
which case the inhibitor comprises an arylamine group that can be
acetylated by NAT. As such, the inhibitor functions as a
competitive inhibitor of the substrate. Suitable inhibitors can be
determined in accordance with the assays set forth in Examples 1
and 2. In addition, wherein the inhibitor is a substrate of NAT, it
is preferred that acetylation of the inhibitor does not result in a
toxic or carcinogenic product. Preferably, the inhibitor of NAT is
a compound of formula: ##STR2##
wherein one or more carbon atoms at positions 2, 3, 5 and 6 can be
heteroatoms, which can be the same or different and can be selected
from the group consisting of oxygen, nitrogen and sulfur, R.sub.1
is one or more substituents, which can be the same or different,
selected from the group consisting of hydrogen, hydroxy, an alkoxy,
sulfhydryl, nitro, amino, a halo, an aryloxy, cyano, --[SO.sub.2
--R.sub.4 ], an alkyl, a cycloalkyl, a heterocycloalkyl, an
alkenyl, an alkynyl, an aryl, a heteroaryl, an arylalkyl, and a
heteroarylalkyl, R.sub.2 is a substituent selected from the group
consisting of amino, carboxyamide, sulfamide, --[NH--NH.sub.2 ],
--[SO.sub.2 --NH--NH.sub.2 ] and --[CO--NH--NH.sub.2 ], and R.sub.3
is a substituent selected from the group consisting of hydrogen,
hydroxy, an alkoxy, sulfhydryl, nitro, amino, a halo, an aryloxy,
cyano, --[SO.sub.2 --R.sub.4 ], an alkyl, a cycloalkyl, a
heterocycloalkyl, an alkenyl, an alkynyl, an aryl, a heteroaryl, an
arylalkyl and a heteroarylalkyl, wherein R.sub.4 is a substituent
selected from the group consisting of hydrogen, hydroxy, an alkoxy,
sulfhydryl, nitro, amino, a halo, an aryloxy, cyano, an alkyl, a
cycloalkyl, a heterocycloalkyl, an alkenyl, an alkynyl, an aryl, a
heteroaryl, an arylalkyl, a heteroarylalkyl, and NH--R.sub.5,
wherein R.sub.5 is a substituent selected from the group consisting
of hydrogen, hydroxy, an alkoxy, sulfhydryl, nitro, amino, a halo,
an aryloxy, cyano, an alkyl, a cycloalkyl, a heterocycloalkyl, an
alkenyl, an alkynyl, an aryl, a heteroaryl, an arylalkyl and a
heteroarylalkyl, wherein the substituent is unsubstituted or
substituted with one to three groups, which can be the same or
different and can be selected from the group consisting of an
alkyl, an alkenyl, an alkynyl, .dbd.O, a halo, hydroxy, a lower
alkoxy, carboxy, a carboalkoxy, a carboxamido, cyano, carbonyl,
--NO.sub.2, an alkylthio, sulfoxide, sulfone, acylamino, amidino,
an aryl, such as phenyl and benzyl, a heteroaryl, an aryloxy, such
as phenoxy and benzyloxy, and a heteroaryloxy.
"Alkyl" includes linear and branched alkyl groups, such as alkyl
groups of twenty carbons or less, preferably from about 1 to about
10 carbon atoms, more preferably from about 1 to about 8 carbon
atoms, and most preferably from about 1 to about 6 carbon atoms.
Examples of such alkyl radicals include methyl, ethyl, propyl,
isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, isoamyl, hexyl,
octyl, dodecanyl, and the like. "Cycloalkyl" includes cyclic alkyl
groups, which comprise a linear and/or branched alkyl group,
preferably twenty carbons or less. Preferably, the cycloalkyl
comprises a cyclic C.sub.3 -C.sub.8 alkyl group.
"Heterocycloalkyl" includes a cyclic alkyl group as described above
in which at least one of the methylene groups is replaced by a
heteroatom, such as a nitrogen or sulfur, which may be
unsubstituted or substituted. Examples of heterocycloalkyls include
tetrahydrofuranyl, piperidine and the like.
"Alkenyl" includes a hydrocarbon of a linear, branched or cyclic
configuration and combinations thereof, which comprises at least
one double bond. Preferably, the alkenyl comprises from about 2 to
about 20 carbon atoms, preferably from about 2 to about 10 carbon
atoms, more preferably from about 2 to about 8 carbon atoms, and
most preferably from about 2 to about 6 carbon atoms. Examples of
alkenyl radicals include vinyl, allyl, 1,4-butadienyl, isopropenyl,
and the like.
"Alkynyl" includes a hydrocarbon of a linear or branched
configuration and combinations thereof, which comprises at least
one triple bond and comprises from about 2 to about 20 carbon
atoms, preferably from about 2 to about 10 carbon atoms, more
preferably from about 2 to about 8 carbon atoms, and most
preferably from about 2 to about 6 carbon atoms. Examples of
alkynyl radicals include ethynyl, propynyl (propargyl), butynyl,
and the like.
"Aryl" and "heteroaryl" include a 5- or 6-membered aromatic or
heteroaromatic ring containing one or more heteroatoms, preferably
no more than three heteroatoms, which are selected from the group
consisting of oxygen, nitrogen and sulfur, a bicyclic 9- or
10-membered aromatic or heteroaromatic ring system containing one
or more heteroatoms, preferably no more than three heteroatoms,
which are selected from the group consisting of oxygen, nitrogen
and sulfur. Examples of aryl groups include, for example, phenyl,
naphthyl, and biphenyl groups. Examples of heteroaryls include, for
example, imidazole, thiophene, and oxazole groups. The aryl and
heteroaryl groups can be substituted with one or more groups,
preferably no more than three groups, selected from the group
consisting of an unsubstituted or a substituted C.sub.1 -C.sub.8
alkyl, an alkenyl, an alkynyl, .dbd.O, --NO.sub.2, halo, hydroxy,
alkoxy, carboxy, a carboalkoxy, a carboxamido, --OCH(COOH).sub.2,
cyano, a carbonyl, an alkylthio, sulfoxide, sulfone, an acylamino,
amidino, an aryl, such as phenyl or benzyl, an aryloxy, such as
phenoxy or benzyloxy, heteroaryl and heteroaryloxy, wherein each of
said aryl, aryloxy, heteroaryl and heteroaryloxy is optionally
substituted with one or more groups, preferably no more than three
groups, which are selected from the group consisting of a C.sub.1
-C.sub.8 alkyl, an alkenyl, an alkynyl, .dbd.O, a halo, a hydroxyl,
an alkoxy, carboxy, a carboalkoxy, a carboxamido, cyano, carbonyl,
an alkylthio, sulfoxide, sulfone, an acylamino, amidino, an aryl,
such as phenyl or benzyl, an aryloxy, such as benzyloxy,
carboxamido, a heteroaryl, a heteroaryloxy and --NO.sub.2. By
"arylalkyl" and "heteroarylalkyl" is meant an aryl or heteroaryl
group additionally comprising a linear or branched alkyl group,
preferably of twenty carbons or less.
The term "alkoxy" as used herein means an alkyl group as defined
herein, wherein at least one alkyl hydrogen atom is replaced by an
oxygen atom. Examples of alkoxy groups include, for example,
methoxy, ethoxy, isopropoxy, and the like. By "aryloxy" and
"heteroaryloxy" is meant an aryl or a heteroaryl group as defined
herein, wherein at least one aryl or heteroaryl hydrogen atom is
replaced by an oxygen atom.
"Halo" can be any suitable halogen. Preferably, "halo" is fluorine,
chlorine, bromine or iodine.
Alkyl, cycloalkyl, heterocycloalkyl, alkenyl and alkynyl can be
unsubstituted or substituted with one or more groups, preferably no
more than three groups, which are selected from the group
consisting of an alkyl, an alkenyl, an alkynyl, .dbd.O, a halo,
hydroxy, a lower alkoxy, preferably of twenty carbons or less,
carboxy, a carboalkoxy, a carboxamido, cyano, carbonyl, --NO.sub.2,
an alkylthio, sulfoxide, sulfone, acylamino, amidino, an aryl, such
as phenyl or benzyl, a heteroaryl, an aryloxy, such as phenoxy or
benzyloxy, and a heteroaryloxy, wherein aryl, heteroaryl, aryloxy
and heteroaryloxy can be substituted as well.
As stated above, there are only 2 forms of human arylamine N-acetyl
transferase, namely, NAT-1 and NAT-2. The inhibitor for use in the
inventive method can be an inhibitor of either NAT-1 or NAT-2 or
both. When the inhibitor inhibits acetylation of a substrate by
NAT-1, preferably the inhibitor is selected from the group
consisting of para-amino-salicylate (PAS) and para-amino-benzoic
acid (PABA). When the inhibitor inhibits the activity of NAT-2, the
inhibitor is preferably selected from the group consisting of
dichlorphenamide, sulphamethazine (SMZ), dapsone (DDS) and
isoniazid (INH).
Compounds of the above formula are widely available commercially.
Those compounds that are not commercially available can be readily
prepared using organic synthesis methods known in the art.
A "substrate" can be any compound comprising an arylamine group
which can be acetylated by NAT and includes substrates that occur
naturally in the body, are ingested, whether in solid or liquid
form as food or supplements, are inhaled, are absorbed through the
skin, or are administered as therapeutic or prophylactic active
agents, e.g., pharmaceutical agents or drugs. Such substrates are
well-known in the art. Examples of suitable substrates include
aminosalicylates, such as 5-aminosalicylate and its prodrug,
sulfasalazine. All sulfonamides are appropriate substrates with the
exception of topical creams such as sulfadoxine, sulfisoxole,
sulfapyridine and sulfaphenazole. Several anticancer drugs are also
acted upon by NAT, such as aminoglutethimide and the
investigational drugs amonafide and batracylin. Substrates of NAT
also include compounds found in the environment, such as substances
in drinking water or tobacco smoke. Such compounds naturally
comprise an arylamine group that can be acetylated by NAT or
comprise an arylamine group that can be acetylated by NAT as a
result of a metabolic process, e.g., an arylamine group that
becomes exposed or is added as a result of a metabolic process.
Substrates of NAT can be determined using the methods set forth in
Example 1, for example, or other suitable methods as are known in
the art.
Oftentimes, acetylation of a drug in vivo adversely affects its
therapeutic or prophylactic benefit. Inhibition of acetylation of a
drug by NAT can increase the drug's therapeutic effect. For
example, inhibiting acetylation of a drug can increase its
effective concentration or prolong its half-life in vivo.
Although metabolism generally lowers the toxicity of drugs, the
opposite effect is often encountered with NAT, i.e., the metabolite
is more toxic than the parent drug. The present inventive method
is, therefore, also useful for reducing the formation of toxic or
carcinogenic metabolites of a drug that is acetylated by NAT. If an
inhibitor is co-administered with a drug in accordance with the
present inventive method, toxicity to the recipient can be
decreased because there will be less exposure to the toxic or
carcinogenic metabolite, and the beneficial effects of the parent
drug can be maximized. The present inventive method can be applied
in many therapeutic areas, since drugs which are metabolized by NAT
are used in most medical disciplines, including heart disease,
infectious diseases, oncology and the like. In the context of the
present inventive methods, "co-administered" is intended to
encompass simultaneous administration of a substrate and an
inhibitor as well as administration in either order and
sufficiently close in time as to realize inhibition of acetylation
of the substrate by NAT.
Heretofore, no one has combined a NAT inhibitor with a compound
comprising an arylamine group that can be acetylated by NAT, such
as a drug, in a single composition, such as a pharmaceutical
composition, in order to enhance the therapeutic effect of the drug
as described above. Accordingly, the present invention also
provides a composition comprising a compound comprising an
arylamine group that can be acetylated by NAT, such as the
substrates described above, and an inhibitor as described above.
The inhibitor interacts with NAT to inhibit NAT from acetylating
the arylamine group in the compound.
For example, a composition can be a water supply comprising a NAT
inhibitor. The inhibitor can block acetylation of compounds known
to be procarcinogenic in contaminated drinking water.
In addition to improving the therapeutic use of a variety of
marketed or investigational drugs, the present inventive method can
be used in the prophylaxis of biological disorders or disease in
which acetylation predisposes an animal to such a biological
disorder or disease. For example, many epidemiological studies have
noted that a high capacity of acetylation by NAT is linked to a
greater incidence of human tumors. As such, the products of
acetylation are carcinogenic. According to the claimed method, an
inhibitor of NAT can be administered prophylactically to reduce
acetylation of a substrate, which, in turn, can lead to a decrease
in a biological disorder in which NAT plays an adverse role, e.g.,
human cancer. One of ordinary skill in the art will appreciate
that, although complete blockage of acetylation is preferred, any
reduction in acetylation will be useful in the prophylaxis of a
biological disorder or disease. Similarly, procarcinogens found in
the environment can be ingested, absorbed through the skin, or
inhaled, i.e., in tobacco smoke. The present invention is useful in
inhibiting the acetylation of such compounds found in the
environment, thereby attenuating the formation of carcinogenic
substances in the body.
In addition to the above-described embodiments, in vitro
embodiments of the present inventive methods also have utility. The
present inventive method of inhibiting acetylation of a substrate
by NAT can be employed for research in numerous disciplines,
including enzymology and clinical pharmacology.
The skilled artisan will appreciate that suitable methods of
administering an inhibitor of NAT, alone or in further combination
with a substrate, e.g., the compositions described herein, are
available. Although more than one route can be used to administer a
particular NAT inhibitor, a particular route can provide a more
immediate and more effective reaction than another route.
Accordingly, the described routes of administration are merely
exemplary and are in no way limiting.
The dose administered to a mammal, particularly a human, in
accordance with the present invention should be sufficient to
effect the desired response in the animal over a reasonable time
frame. One skilled in the art will recognize that dosage will
depend upon a variety of factors, including the strength of the
particular substrate and NAT inhibitor employed, as well as the
age, species and body weight of the animal. The size of the dose
also will be determined by the route, timing and frequency of
administration as well as the existence, nature, and extent of any
adverse side effects that might accompany the administration of a
particular substrate and NAT inhibitor and the desired
physiological effect.
Suitable doses and dosage regimens can be determined by
conventional range-finding techniques known to those of ordinary
skill in the art. Dosages for some inhibitors suitable for use in
the present invention are known to those of skill in the art as
they are widely administered to patients for indications other than
inhibition of acetylation of a substrate.
One of ordinary skill in the art will appreciate that the form the
composition comprising a compound comprising an arylamine and a NAT
inhibitor will take is determined by the particular compound (e.g.,
drug) and particular inhibitor (e.g., inhibitor comprising an
arylamine group) used. Determination of appropriate combinations of
compound and inhibitor is well within the skill in the art and,
furthermore, can be determined using the methods set forth in
Examples 1-3. Preferably, acetylation of the inhibitor does not
result in a toxic or carcinogenic product.
Compositions for use in the present inventive method preferably
comprise a carrier, such as a pharmaceutically acceptable carrier,
and an amount of a NAT inhibitor sufficient to inhibit acetylation
of a substrate. The carrier can be any of those conventionally used
and is limited only by chemico-physical considerations, such as
solubility and lack of reactivity with the compound, and by the
route of administration. It will be appreciated by one of ordinary
skill in the art that, in addition to the following described
pharmaceutical compositions, the NAT inhibitor can be formulated as
polymeric compositions, inclusion complexes, such as cyclodextrin
inclusion complexes, liposomes, microspheres, microcapsules and the
like.
The pharmaceutically acceptable excipients described herein, for
example, vehicles, adjuvants, carriers or diluents, are well-known
to those who are skilled in the art and are readily available to
the public. It is preferred that the pharmaceutically acceptable
carrier be one which is chemically inert to the NAT inhibitor and
one which has no detrimental side effects or toxicity under the
conditions of use.
The choice of excipient will be determined in part by the
particular NAT inhibitor, as well as by the particular method used
to administer the composition. Accordingly, there is a wide variety
of suitable formulations of the pharmaceutical composition of the
present invention. The following formulations are merely exemplary
and are in no way limiting.
Injectable formulations are among those that are preferred in
accordance with the present inventive method. The requirements for
effective pharmaceutical carriers for injectable compositions are
well-known to those of ordinary skill in the art (see Pharmaceutics
and Pharmacy Practice, J. B. Lippincott Co., Philadelphia, Pa.,
Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook
on Injectable Drugs, Toissel, 4.sup.th ed., pages 622-630 (1986)).
It is preferred that such injectable compositions be administered
intramuscularly, subcutaneously, intravenously, or
intraperitoneally.
Topical formulations are well-known to those of skill in the art.
Formulations suitable for topical administration may be presented
as creams, gels, pastes, or foams, containing, in addition to the
active ingredient, such carriers as are known in the art to be
appropriate. Such formulations are suitable in the context of the
present invention for application to the skin.
Formulations suitable for oral administration can consist of (a)
liquid solutions, such as an effective amount of the compound
dissolved in diluents, such as water, saline, or orange juice; (b)
capsules, sachets, tablets, lozenges, and troches, each containing
a predetermined amount of the active ingredient, as solids or
granules; (c) powders; (d) suspensions in an appropriate liquid;
and (e) suitable emulsions. Liquid formulations may include
diluents, such as water and alcohols, for example, ethanol, benzyl
alcohol, and the polyethylene alcohols, either with or without the
addition of a pharmaceutically acceptable surfactant, suspending
agent, or emulsifying agent. Capsule forms can be of the ordinary
hard- or soft-shelled gelatin type containing, for example,
surfactants, lubricants, and inert fillers, such as lactose,
sucrose, calcium phosphate, and corn starch. Tablet forms can
include one or more of lactose, sucrose, mannitol, corn starch,
potato starch, alginic acid, microcrystalline cellulose, acacia,
gelatin, guar gum, colloidal silicon dioxide, croscarmellose
sodium, talc, magnesium stearate, calcium stearate, zinc stearate,
stearic acid, and other excipients, colorants, diluents, buffering
agents, disintegrating agents, moistening agents, preservatives,
flavoring agents, and pharmacologically compatible excipients.
Lozenge forms can comprise the active ingredient in a flavor,
usually sucrose and acacia or tragacanth, as well as pastilles
comprising the active ingredient in an inert base, such as gelatin
and glycerin, or sucrose and acacia, emulsions, gels, and the like
containing, in addition to the active ingredient, such excipients
as are known in the art.
Aerosol formulations to be administered via inhalation are also
appropriate. These aerosol formulations can be placed into
pressurized acceptable propellants, such as
dichlorodifluoromethane, propane, nitrogen, and the like. They also
can be formulated as pharmaceuticals for non-pressured preparations
such as in a nebulizer or an atomizer.
Formulations suitable for parenteral administration include aqueous
and non-aqueous, isotonic sterile injection solutions, which can
contain anti-oxidants, buffers, bacteriostats, and solutes that
render the formulation isotonic with the blood of the intended
recipient, and aqueous and non-aqueous sterile suspensions that can
include suspending agents, solubilizers, thickening agents,
stabilizers, and preservatives. The inhibitor can be administered
in a physiologically acceptable diluent in a pharmaceutical
carrier, such as a sterile liquid or mixture of liquids, including
water, saline, aqueous dextrose and related sugar solutions, an
alcohol,. such as ethanol, isopropanol, or hexadecyl alcohol,
glycols, such as propylene glycol or polyethylene glycol,
dimethylsulfoxide, glycerol ketals, such as
2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, such as
poly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester
or glyceride, or an acetylated fatty acid glyceride, with or
without the addition of a pharmaceutically acceptable surfactant,
such as a soap or a detergent, suspending agent, such as pectin,
carbomers, methylcellulose, hydroxypropylmethylcellulose, or
carboxymethylcellulose, or emulsifying agents and other
pharmaceutical adjuvants.
Suitable soaps for use in parenteral formulations include fatty
alkali metals, ammonium, and triethanolamine salts, and suitable
detergents include (a) cationic detergents such as, for example,
dimethyl dialkyl ammonium halides, and alkyl pyridinium halides,
(b) anionic detergents such as, for example, alkyl, aryl, and
olefin sulfonates, alkyl, olefin, ether, and monoglyceride
sulfates, and sulfosuccinates, (c) nonionic detergents such as, for
example, fatty amine oxides, fatty acid alkanolamides, and
polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents
such as, for example, alkyl-p-aminopropionates, and
2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures
thereof.
The parenteral formulations may contain preservatives and buffers.
In order to minimize or eliminate irritation at the site of
injection, such compositions may contain one or more nonionic
surfactants having a hydrophile-lipophile balance (HLB) of from
about 12 to about 17.
The quantity of surfactant in such formulations will typically
range from about 5 to about 15% by weight. Suitable surfactants
include polyethylene sorbitan fatty acid esters, such as sorbitan
monooleate and the high molecular weight adducts of ethylene oxide
with a hydrophobic base, formed by the condensation of propylene
oxide with propylene glycol. The parenteral formulations can be
presented in unit-dose or multi-dose sealed containers, such as
ampules and vials, and can be stored in a freeze-dried
(lyophilized) condition requiring only the addition of the sterile
liquid excipient, for example, water, for injections, immediately
prior to use. Extemporaneous injection solutions and suspensions
can be prepared from sterile powders, granules, and tablets of the
kind previously described.
The inhibitor of NAT and a substrate, preferably a drug, can be
co-administered to inhibit acetylation of the substrate. The
present inventive method also can involve the co-administration of
other pharmaceutically active compounds. By "co-administration" is
meant administration before, concurrently with, e.g., in
combination with the NAT inhibitor in the same formulation or in
separate formulations, or after administration of a NAT inhibitor
as described above. For example, vitamins and minerals, e.g., zinc,
anti-oxidants, e.g., carotenoids (such as a xanthophyll carotenoid
like zeaxanthin or lutein), and micronutrients can be
co-administered. One of ordinary skill in the art will appreciate
that administration of vitamins and minerals will be particularly
useful in situations wherein the present inventive method is
employed as a prophylactic measure.
EXAMPLES
The following examples further illustrate the present invention
but, of course, should not be construed as in any way limiting its
scope.
Example 1
This example demonstrates that PAS and PABA are effective
inhibitors of acetylation by NAT-1.
Subcellular Preparations
Human liver was homogenized and prepared by high-speed differential
centrifugation. A preliminary centrifugation was performed at
13,000 g for 20 min. The cytosolic fraction was collected as the
supernatant from a second centrifugation at 105,000 g for 60 min
(105S). Upon the addition of the single cofactor for NAT, acetyl
co-enzyme A, and its regenerating system, 1 mM acetyl-L-carnitine
and 1 unit/ml of carnitine-acetyl transferase, the reaction
proceeded following addition of the substrate, i.e., PAS, PABA or
SMX, to the cytosol. Fifteen minutes at 37.degree. C. was
sufficient to generate measurable metabolites. The reaction was
terminated by the addition of perchloric acid to a final
concentration of 1.5%. Parallel incubations were conducted for drug
alone versus drug plus potential inhibitor. Percent inhibition was
calculated from the ratio of [Acetylated metabolite]/[parent] with
and without the potential inhibitor.
The formation of N-acetylated metabolites was quantified by HPLC.
Samples from the incubates were injected onto a reverse-phase
column (e.g., Waters Symmetry C18, 5 micron, 4.6.times.150 mm). The
mobile phase consisted of 1% acetic acid, with a gradient in
acetonitrile from 7% to 19% over 16 minutes, then 19% to 37% over
10 minutes at 1 ml/min. Drugs and their N-acetylated metabolites
were quantitated by UV absorption using a diode-array detector.
PAS incubation with human liver subcellular preparations as
described above resulted in a single metabolic peak. Based upon
retention time and UV spectrum, the peak was identified as Ac-PAS,
an acetylated metabolite. Similar results were obtained for SMX as
illustrated by FIG. 1, which is a graph of absorbance at 268 nm vs.
time (min). FIG. 1 shows the HPLC results for PAS and SMX, along
with their acetylated metabolites, Ac-PAS and Ac-SMX.
The substrates assayed above were then co-incubated with human
liver subcellular preparations. Co-incubation of 200 .mu.M of PAS
with either 20 .mu.M of PABA or 200 .mu.M SMX resulted in blockage
of all measurable acetylation to Ac-PABA and 50% inhibition of
acetylation to Ac-SMX. Similarly, when 200 .mu.M of PABA was
co-incubated with either 20 .mu.M of PAS or 200 .mu.M of SMX, no
Ac-PAS was detected and Ac-SMX formation was inhibited by 50%.
Hepatocyte Culture
Human hepatocytes were incubated at 37.degree. C. at a ratio of 1
million cells per ml in modified Williams E buffer. Either
cryopreserved hepatocytes in suspension or freshly-isolated
hepatocytes attached to microtiter plates were used. The reaction
was initiated by addition of substrate, and terminated after 4
hours by the addition of perchloric acid. As before, parallel
incubations were conducted for drug alone versus drug plus
potential inhibitors. Percent inhibition was calculated from the
ratio of [Acetylated metabolite]/[parent] with and without the
potential inhibitor.
Incubation of 200 .mu.M of PAS or 200 .mu.M of SMX separately with
hepatocytes resulted in formation of Ac-PAS and Ac-SMX. When
co-incubated, the same amount of Ac-PAS was found, but Ac-SMX was
reduced by 60%.
The preceding results demonstrate that both PAS and PABA are
effective competitive inhibitors of the acetylation of SMX by
NAT-1.
Example 2
This example demonstrates that dichlorphenamide, sulphamethazine
(SMZ), dapsone (DDS) and isoniazid (INH) are effective inhibitors
of acetylation by NAT-2.
Subcellular Preparations
INH, DDS, SMZ were incubated with human liver subcellular
preparations as described in Example 1. The reactions were allowed
to proceed for thirty minutes at 37.degree. C. before termination
by addition of perchloric acid. Parallel incubations were conducted
for drug alone versus drug plus potential inhibitors, namely
dichlorphenamide, SMZ, INH and aminoglutethimide (AG). The
formation of N-acetylated metabolites was quantified by HPLC. For
INH, 1-octanesulfonic acid (5 mM) was added to the mobile
phase.
Acetylated metabolites were found when INH, DDS or SMZ were
incubated separately with cytosol. The cross inhibition with each
other and by dichlorphenamide are shown in Table I.
TABLE I Inhibitors Substrate dichlorphenamide SMZ INH AG DDS ++ ++
++ (N/A) SMZ ++ (N/A) ++ INH -- (N/A) (N/A) ++ = strong inhibition
-- = no inhibition (N/A) = not tested
Hepatocyte Culture
INH, DDS and SMZ were incubated with human hepatocytes as described
in Example 1. Parallel cultures were conducted for each drug alone
and each drug plus dichlorphenamide as a potential inhibitor. An
additional culture of DDS was incubated with SMZ, a potential
inhibitor. Dichlorphenamide strongly inhibited the acetylation of
all substrates. In addition, SMZ inhibited acetylation of DDS.
The preceding results illustrate that dichlorphenamide, SMZ, DDS,
and INH are effective inhibitors of NAT-2.
Example 3
This example demonstrates that PAS inhibits the acetylation of SMX,
when PAS and SMX are administered in vivo at their standard
doses.
Based upon the in vitro results reported in Examples 1 and 2,
clinical inhibition studies of acetylation were designed. SMX was
given to a volunteer human subject at its standard dose of 500 mg
every 12 hours, for a total of 5 days. On the fourth and fifth
days, PAS was also administered at its standard dose of 4 g every 8
hours. Plasma and urine concentrations of SMX and its acetylated
metabolite, Ac-SMX, were measured on the third and fifth day of
dosing. Day 3 represents baseline pharmacokinetics of SMX and
Ac-SMX, while Day 5 demonstrates the impact of PAS upon blocking
the formation of Ac-SMX.
As shown in FIG. 2, which is a graph of the plasma concentration of
Ac-SMX (mM) vs. time (hours), shows that substantial concentrations
of Ac-SMX were observed in plasma on Day 3, but were almost
completely abolished on Day 5, indicating essentially complete
inhibition of metabolism of SMX to form Ac-SMX. In addition,
substantial amounts of Ac-SMX were excreted into urine on Day 3,
but not on Day 5, further reflecting successful inhibition by PAS
of SMX acetylation, as illustrated in FIG. 3, which is a graph of
urine concentration of Ac-SMX (mM) vs. time (hours).
Since acetylation is one of the major pathways for SMX elimination,
blockage of that pathway produced higher plasma concentrations of
SMX on Day 5 than on Day 3, as exemplified by FIG. 4, which is a
graph of plasma concentration of SMX (mM) vs. time (hours).
At the conventional doses used in this study, the PAS
concentrations in plasma, which were measured on Day 5 using HPLC
as shown in FIG. 5, which is a graph of plasma concentration of PAS
(mm) vs. time (hours), were found to exceed the levels required to
inhibit NAT-1 in the screening experiments with human liver
tissue.
This example exemplifies the ability of an inhibitor of NAT to
block metabolism of a drug in vivo.
All of the references cited herein, including patents, patent
applications, and publications, are hereby incorporated in their
entireties by reference.
While this invention has been described with an emphasis upon
preferred embodiments, it will be obvious to those of ordinary
skill in the art that variations of the preferred embodiments may
be used and that it is intended that the invention may be practiced
otherwise than as specifically described herein. Accordingly, this
invention includes all modifications encompassed within the spirit
and scope of the invention as defined by the following claims.
* * * * *